Pharmaceutical compositions comprising unacylated ghrelin and therapeutical uses thereof

ABSTRACT

The present invention relates to compositions containing unacylated ghrelin and derivatives thereof and their uses in the control of glycemia in ageing patients, GH deficient patients, diabetic patients and obese patients.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/756,456 filed on May 31, 2007 which is acontinuation-in-part of U.S. patent application Ser. No. 10/499,376,which has the international filing date of Dec. 18, 2002, and enteredthe U.S. national phase on Nov. 29, 2004, which U.S. patent applicationclaims the benefit of priority of Canadian Patent Application No.2,365,704, filed Dec. 18, 2001, the contents of which are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to new compositions comprising unacylated ghrelinand their therapeutical uses thereof.

2. Description of Prior Art

Ghrelin is a recently discovered gastric hormone of 28 amino acidsshowing a unique structure with an n-octanoyl ester at its third serineresidue (Kojima M et al. Nature 1999; 402(6762):656660). Though manysynthetic peptidyl and nonpeptidyl growth hormone (GH) secretagogues(GHS) were identified as ligands of GHS-R, ghrelin is shown to be aphysiological ligand for the GHS-R. Ghrelin powerfully stimulates GHsecretion through its interaction with GHS-R both in animals and inhumans (Ukkola, O et al., 2002 Ann. Med. 34:102-108). The GH-releasingactivity of ghrelin is mediated by activation of GHS-R at the pituitaryand, mainly, at the hypothalamic level (Kojima M et al. Nature 1999;402(6762):656660) likely by enhancing the activity of growth hormonereleasing hormone (GHRH)-secreting neurons and, concomitantly, acting asa functional somatostatin (SS) antagonist (Ghigo E et al. Eur JEndocrinol 1997; 136(5):445460). Other mechanisms have been postulatedrecently as well (Ahnfelt-Ronne I et al. Endocrine 2001; 14(1):133-135).The interplay among various factors leading to GH secretion is depictedin FIG. 1.

The GHS-R and its subtypes are not restricted to thehypothalamus-pituitary unit but are present also in other central andperipheral tissues (Papotti M et al. J Clin Endocrinol Metab 2000;85(10):3803-3807) and the physiological actions of ghrelin, as well asthose of synthetic GHS are not restricted to GH secretion. In fact,ghrelin stimulates lactotroph and corticotroph hormone secretion, hasorexigenic and cardiovascular actions, shows anti proliferative effectson thyroid and breast tumors and regulates gastric motility and acidsecretion through vagal mediation (Ukkola, O et al., 2002, Ann. Med.34:102-108).

In humans, fasting leads to elevated serum GH concentrations.Traditionally, changes in hypothalamic GHRH and somatostatin have beenconsidered as the main mechanisms, which induce elevations in GHsecretion during fasting. As ghrelin administration in man alsostimulates GH release, and serum ghrelin concentrations are elevatedduring fasting, increased ghrelin actions might be another mechanismwhereby fasting results in the stimulation of GH release.

Although ghrelin is likely to regulate pituitary GH secretion ininterplay with GHRH and SS, GHS receptors have also been identified onhypothalamic neurons and in the brainstem (Nakazato M et al. Nature2001; 409(6817):194-198). Apart from potential paracrine effects,ghrelin may thus offer an endocrine link between the stomach,hypothalamus and pituitary, suggesting an involvement in the regulationof energy balance. Tschop et al. have shown that daily peripheraladministration of ghrelin in mice and rats caused weight gain byreducing fat utilization (Tschop M et al. Nature 2000; 19;407(6806):908-913). Intracerebroventricular administration of ghrelingenerated a dose dependent increase in food intake and body weight. Ratserum ghrelin concentrations increased by fasting and decreased byre-feeding or oral glucose administration, but not by water ingestion.Apparently ghrelin, in addition to its role in regulating GH secretion,signals the hypothalamus when an increase in metabolic efficiency isnecessary (Tschop M et al. Nature 2000; 19; 407(6806):908-913; Muller AF et al. Clin Endocrnol (Oxf) 2001; 55(4):461-467).

Studies by Kojima and others have shown that unacylated ghrelin (UAG)has no affinity to the known GHS-R (GHS-R1a receptor), which isresponsible for GH release from the pituitary gland (Kojima M et al.Nature 1999; 402(6762):656-660). This was confirmed later by Bednarek MA et al (Bednarek M A et al, J. Med Chem. 2000, 43:4370-4376), whoshowed that unacylated ghrelin could not be a physiological ligand ofthe GHS-R1a receptor (IC₅₀>10,000 nM), since it poorly activated GHS-R1aat micromolar concentrations; large hydrophobic acyl group is obligatoryat position 3 of ghrelin for its biological response on GH secretion.

The PCT application, WO 01/87335A2, discloses methods of selectivelyinhibiting ghrelin actions including those on obesity using growthhormone secretagogue receptor antagonists and ghrelin neutralizingreagents. The ghrelin neutralizing reagents are antibodies, single chainantibodies, antibody fragments, or antibody-based constructs.

Specific binding of acylated ghrelin can be found in many peripheraltissues (Papotti M et al. J Clin Endocrinol Metab 2000;85(10):3803-3807). In these tissues, no mRNA expression of the GHS-R1areceptor could be found, indicating that other receptor (sub)types ofreceptors that can bind GHS would be responsible for this specificbinding. These novel receptors may mediate ghrelin's peripheral actionswhich are, as shown in this invention, efficiently antagonized byunacylated ghrelin. These novel receptors may also mediate unacylatedghrelin direct actions on metabolism and cell proliferation, as shown inthe present invention.

It would be highly desirable to be provided with pharmaceuticalcompositions of nonacylated ghrelin for glycemic control in certainmetabolic diseases and disorders and methods to treat them.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method forpreventing and/or treating a metabolic disorder associated with impairedglucose metabolism in a patient comprising administering atherapeutically effective amount of an agent selected from the groupconsisting of an unacylated ghrelin, an analog thereof and apharmaceutically acceptable salt thereof, to said patient.

In accordance with the present invention, there is provided a method asdescribed above, wherein treatment of the metabolic disorder associatedwith impaired glucose metabolism is through enhancement of proliferationor of survival of insulin-secreting cells.

In accordance with the present invention, there is provided a method asdescribed above, wherein the enhancement of proliferation or survival ofinsulin-secreting cells is achieved by administration of the unacylatedghrelin or the analog thereof in the patient.

In accordance with the present invention, there is provided a method asdescribed above, wherein the enhancement of survival is achieved ex vivoby subjecting the insulin-secreting cells to the unacylated ghrelin orthe analog thereof prior to administering said cells to the patient as agraft.

In accordance with the present invention, there is provided a method forenhancing survival and/or proliferation of insulin-secreting cellscomprising culturing said cells in the presence of a therapeuticallyeffective amount of an agent selected from the group consisting ofunacylated ghrelin and an analog thereof.

In accordance with the present invention, there is provided a method forinhibiting death of insulin-secreting cells comprising culturing saidcells in the presence of a therapeutically effective amount of an agentselected from the group consisting of unacylated ghrelin and an analogthereof.

In accordance with the present invention, there is provided acomposition for preventing and/or reducing postprandial induction ofinsulin resistance comprising a therapeutically effective amount of atleast one of unacylated ghrelin, an analog thereof and apharmaceutically acceptable salt thereof in association with apharmaceutically acceptable carrier.

The composition in accordance with a preferred embodiment of the presentinvention, wherein the unacylated ghrelin is having an amino acid as setforth in SEQ ID NO: 1.

In accordance with the present invention, there is provided a method forreducing postprandial induction of insulin resistance in a patientcomprising the step of administering a therapeutically effective amountof the composition of the present invention to the patient.

The method in accordance with a preferred embodiment of the presentinvention, wherein the administration is through a route selected fromthe group consisting of intravenous, subcutaneous, transdermal, oral,buccal, sublingual, nasal and by inhalation.

The method in accordance with a preferred embodiment of the presentinvention, wherein the composition is administered in a dose varyingfrom about 0.001 μg/kg to about 10.0 mg/kg, more preferably from about 1μg/kg to about 1 mg/kg.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for reducing postprandialinduction of insulin resistance in a patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for reducing postprandial induction of insulin resistance ina patient.

In accordance with the present invention, there is provided acomposition for preventing and/or reducing dawn phenomenon in type Idiabetes patient comprising a therapeutically effective amount of atleast one of unacylated ghrelin, an analog thereof and apharmaceutically acceptable salt thereof in association with apharmaceutically acceptable carrier.

In accordance with the present invention, there is provided a method forpreventing and/or reducing dawn phenomenon in type I diabetes patientcomprising the step of administering a therapeutically effective amountof the composition of the present invention to the patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for preventing and/or reducingdawn phenomenon in type I diabetes patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for preventing and/or reducing dawn phenomenon in type Idiabetes patient.

In accordance with the present invention, there is provided acomposition for reducing body weight increased in a patient sufferingfrom at least one of type II diabetes and syndrome X comprising atherapeutically effective amount of at least one of unacylated ghrelin,an analog thereof, and a pharmaceutically acceptable salt thereof inassociation with a pharmaceutically acceptable carrier.

The composition in accordance with a preferred embodiment of the presentinvention, wherein the patient is treated with oral antidiabetic drugs.

In accordance with the present invention, there is provided a method forreducing a body weight increased encountered by a patient suffering fromat least one of type II diabetes and syndrome X comprising the step ofadministering a therapeutically effective amount of the composition ofthe present invention.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for reducing a body weightincreased encountered by a patient suffering from at least one of typeII diabetes and syndrome X.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for reducing a body weight increased encountered by a patientsuffering from at least one of type II diabetes and syndrome X.

In accordance with the present invention, there is provided acomposition for facilitating treatment of an insulin-resistant patientcomprising a therapeutically effective amount of at least one ofunacylated ghrelin, an analog thereof and a pharmaceutically acceptablesalt thereof in association with a pharmaceutically acceptable carrier.

In accordance with the present invention, there is provided a method forfacilitating the treatment of an insulin-resistant patient comprisingthe step of administering a therapeutically effective amount of thecomposition of the present invention to the patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for facilitating the treatmentof an insulin-resistant patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for facilitating treatment of insulin-resistant patient.

In accordance with the present invention, there is provided acomposition for decreasing fat mass in a growth hormone-deficientpatient comprising a therapeutically effective amount of at least one ofunacylated ghrelin, an analog thereof and a pharmaceutically acceptablesalt thereof in association with a pharmaceutically acceptable carrier.

In accordance with the present invention, there is provided a method fordecreasing fat mass in a growth hormone-deficient patient comprising thestep of administering a therapeutically effective amount of thecomposition of the present invention to the patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for decreasing fat mass in agrowth hormone-deficient patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for decreasing fat mass in a growth hormone-deficientpatient.

In accordance with the present invention, there is provided acomposition for decreasing fat mass in an ageing patient having a highbody mass index comprising a therapeutically effective amount of atleast one of unacylated ghrelin, an analog thereof and apharmaceutically acceptable salt thereof in association with apharmaceutically acceptable carrier.

In accordance with the present invention, there is provided a method fordecreasing fat mass in an ageing patient having a high body mass indexcomprising the step of administering a therapeutically effective amountof the composition of the present invention to the patient.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for decreasing fat mass in anageing patient having a high body mass index.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for decreasing fat mass in an ageing patient having a highbody mass index.

In accordance with the present invention, there is provided acomposition for preventing and/or reducing insulin resistance in apatient comprising a therapeutically effective amount of at least one ofunacylated ghrelin, an analog thereof and a pharmaceutically acceptablesalt thereof in association with a pharmaceutically acceptable carrier.

In accordance with the present invention, there is provided a method forpreventing and/or reducing insulin resistance in a patient in severecatabolism comprising the step of administering to said patient atherapeutically effective amount of the composition of the presentinvention.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for preventing and/or reducinginsulin resistance in a patient in severe catabolism.

In accordance with the present invention, there is provided the use ofthe composition of the present invention for the preparation of amedicament for preventing and/or reducing insulin resistance in apatient in severe catabolism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the interplay among various factors leading to GHsecretion;

FIG. 2A illustrates glucose concentration over time in absence and inpresence of the GH receptor antagonist pegvisomant upon administrationof GHRP-6;

FIG. 2B illustrates insulin concentration over time in absence and inpresence of the GH receptor antagonist pegvisomant upon administrationof GHRP-6;

FIG. 3 illustrates the serum Ghrelin, and GH concentrations (Solid line:ghrelin levels; dotted line: GH levels) during fasting for three daysand after a bolus injection of GHRP-6 on day 4;

FIG. 4A illustrates insulin variation over time in a patient havingreceived a single intravenous administration of human ghrelin (soliddots) or placebo (open dots);

FIG. 4B illustrates glucose variation over time in a patient havingreceived a single intravenous administration of human ghrelin (soliddots) or placebo (open dots);

FIG. 5A illustrates glucose level over time in a patient having beenadministered with ghrelin, desoct-ghrelin or ghrelin and desoct-ghrelin;

FIG. 5B illustrates insulin level over time in a patient having beenadministered with ghrelin, desoct-ghrelin or ghrelin and desoct-ghrelin;

FIGS. 6A and 6B illustrate the free fatty acid (FFA) profile and AUCduring saline or unacylated ghrelin infusion (1.0 μg/Kg/h, from 21.00 to13.00 h) in healthy subjects;

FIGS. 6C and 6D illustrate glucose profile and AUC during saline orunacylated ghrelin infusion (1.0 μg/Kg/h, from 21.00 to 13.00 h) inhealthy subjects;

FIG. 6E illustrates insulin and FIG. 6F illustrates glucagon profileduring saline or unacylated ghrelin infusion (1.0 μg/Kg/h, from 21.00 to13.00 h) in healthy subjects;

FIG. 6G illustrates growth hormone (GH) and FIG. 6H illustrates cortisolprofile during saline or unacylated ghrelin infusion (1.0 μg/Kg/h, from21.00 to 13.00 h) in healthy subjects;

FIG. 7 illustrates a competition for radiolabeled unacylated ghrelin([¹²⁵I-Tyr⁴]-unacylated ghrelin) to HIT-T15 cell membranes by theindicated competitors. Binding is expressed as percentage of control(specific binding in the absence of unlabeled competitor);

FIG. 8A illustrates HIT-T15 cell proliferation in the presence ofincreasing concentration of unacylated ghrelin;

FIG. 8B illustrates HIT-T15 cell survival in the presence of increasingconcentration of unacylated ghrelin;

FIG. 8C illustrates phase-contrast images of cells cultured in thepresence or absence of unacylated ghrelin;

FIG. 8D illustrates effect of unacylated ghrelin on HIT-T15 cellproliferation in the presence of NF499 or pertussis toxin (PTX);

FIG. 8E illustrates the effect of unacylated ghrelin on HIT-T15 cellviability in the presence of NF499 or pertussis toxin (PTX);

FIG. 9A illustrates a Hoechst 33258 nuclear immunofluorescence stainingof serum starved cells±unacylated ghrelin (upper panel) and cellstreated with IFN-γ/TNF-α±unacylated ghrelin (lower panel);

FIG. 9B illustrates the effect of unacylated ghrelin on apoptosisinduced by serum starvation and IFN-γ/TNF-α synergism in HIT-T15 cells;

FIG. 9C illustrates the effect of unacylated ghrelin on cellproliferation of HIT-T15 cells induced by serum starvation andIFN-γ/TNF-α synergism;

FIG. 9D illustrates unacylated ghrelin secretion in HIT-T15 cellsfollowing exposure to either serum, SF±cytokines;

FIG. 9E illustrates a Hoechst 33258 of cells cultured for 48 h in thepresence of serum or in SF medium alone with addition of an anti-ghrelinantibody;

FIG. 10A illustrates the effect of unacylated ghrelin stimulation forthe indicated time, of intracellular cAMP concentration in HIT-T15cells; and

FIG. 10B illustrates the effect of unacylated ghrelin on cAMP levels inHIT-T15 cells incubated in the presence of serum or in serum-free (SF)medium±unacylated ghrelin alone or with IFN-γ/TNF-α combination.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention belongs.

In the present application, the terms “ghrelin” and “acylated ghrelin”are used interchangeably and are intended to mean the same.

For the purpose of the present invention the following terms are definedbelow.

The term “unacylated ghrelin” is intended to mean peptides that containthe amino acid sequence specified in SEQ ID NO: 1. Naturally-occurringvariations of unacylated ghrelin include peptides that containsubstitutions, additions or deletions of one or more amino acids whichresult due to discrete changes in the nucleotide sequence of theencoding ghrelin gene or its alleles thereof or due to alternativesplicing of the transcribed RNA. It is understood that the said changesdo not substantially affect the antagonistic properties, pharmacologicaland biological characteristics of unacylated ghrelin variant. Thosepeptides may be in the form of salts, particularly the acidic functionsof the molecule may be replaced by a salt derivative thereof such as atrifuoroacetate salt.

The term “analogue of unacylated ghrelin” refers to both structural andfunctional analogues of unacylated ghrelin which are capable ofreplacing unacylated ghrelin in antagonizing the peripheral actions ofghrelin or in providing the same metabolic and cell proliferationeffects as unacylated ghrelin. Simple structural analogues comprisepeptides showing homology with unacylated ghrelin as set forth in SEQ IDNO: 1 or a fragment thereof. For example, an isoform of ghrelin-28 (SEQID NO: 1), des Gln-14 Ghrelin (a 27 amino acid peptide possessing serine3 modification by n-octanoic acid) is shown to be present in stomach; itis functionally identical to ghrelin in that it binds to GHS-R1a withsimilar binding affinity, elicits Ca²⁺ fluxes in cloned cells andinduces GH secretion with similar potency as Ghrelin-28.

The term “homology” refers to sequence similarity between two peptideswhile retaining an equivalent biological activity. Homology can bedetermined by comparing each position in the aligned sequences. A degreeof homology between amino acid sequences is a function of the number ofidentical or matching amino acids at positions shared by the sequencesso that an “homologous sequence” refers to a sequence sharing homologyand an equivalent function or biological activity.

It is known that des-Gln14-ghrelin is a structural analogue and afunctional analogue of ghrelin; as such, unacylated des-Gln14-ghrelincould potentially antagonize effects of ghrelin and des-Gln14-ghrelin onperipheral metabolism involving insulin secretion and glycemic control,or provide the same direct biological effects as unacylated ghrelin onmetabolism or cell proliferation and survival.

Functional analogues of unacylated ghrelin despite their diversity havethe common interesting property of being able to fully replaceunacylated ghrelin in one or more biological activities exhibited byunacylated ghrelin. For example, these biological activities ofunacylated ghrelin may include, binding to a specific receptor, alteringthe signals arising from the activation of said receptor, modulating thefunctional consequences of activation of said receptor.

Functional analogues of unacylated ghrelin, as well as those ofunacylated des-Gln14-ghrelin, are able to produce the biological effectsof unacylated ghrelin, as described in the present application, hencesuch functional analogues will be useful for realizing therapeuticbenefits in medical conditions involving GH-deficient states.

Conservative substitutions of one or more amino acids in the primarysequence of unacylated ghrelin may provide structural analogues of thepeptide. In order to derive more potent analogues, it is customary touse alanine scans, selective substitutions with D-amino acid orsynthetic amino acids, truncation of the peptide sequence in order tofind a “functional core” of the peptide, covalent addition of moleculesto improve the properties of the peptide such as its serum stability, invivo half life, potency, hydrophilicity or hydrophobicity andimmunogenicity.

General methods and synthetic strategies used in providing functionaland structural analogues of peptides is described in publications suchas “Solid phase peptide synthesis” by Stewart and Young, W. h Freeman &Co., San Francisco, 1969 and Erickson and Merrifield, “The Proteins ”Vol. 2, p. 255 et seq. (Ed. Neurath and Hill), Academic Press, New York,1976.

All documents referred to herein are hereby incorporated by reference.

In accordance with the present invention, there is providedpharmaceutical compositions for acting on insulin levels and glycemia inmetabolic diseases and disorders and methods to prevent, reduce andtreat them.

It has been demonstrated that the growth hormone secretagogue, GHRP-6,has direct and non-GH dependent actions on metabolism (Muller A F et al.J Clin Endocrinol Metab 2001; 86(2):590-593). It is shown in the presentapplication that in normal human beings, preprandial GHS administration(1 μg/Kg i.v.) induces a postprandial increase in serum glucose levels,but only in the presence of the GH receptor antagonist pegvisomant (FIG.2: left panel).

Moreover, this is accompanied by an impressive increase in serum insulinconcentrations (indicating insulin resistance; FIG. 2A). TheseGHS-mediated changes indicate that when GH bioactivity is lowered (asseen in GH deficient, ageing, obese and diabetic individuals), GHS caninduce potent changes in metabolic control, which are characteristic ofthe “metabolic syndrome X”. Because in this study GH-action was blockedby pegvisomant, these GHS-mediated metabolic changes on the“gastro-entero-hepatic axis” must be direct and non-pituitary mediated.Supporting this hypothesis, daily ghrelin administration in rodents foronly several days, indeed induces an obese state, again indicating thatthese GHS-mediated effects on metabolism are powerful and clinicallyrelevant.

The data presented in the present application indicate that GHS-mediatedeffects are involved in the induction of the metabolic alterations, aswell as subsequent changes in body composition, which are characteristicfor the insulin resistance syndrome (metabolic syndrome), as observed inGH deficiency, but also during normal ageing, obesity and diabetes.

In order to understand the diurnal rhythms of ghrelin and GH secretionduring fasting, a study was conducted on 10 healthy human volunteerswith normal body mass index. FIG. 3 shows the serum Ghrelin, and GHconcentrations (Solid line: ghrelin levels; dotted line: GH levels)during fasting for three days and after a bolus injection of GHRP-6 onday 4. Fasting rapidly induces a diurnal ghrelin rhythm that is followedby a similar GH rhythm. Administration of 1 μg/kg of GHRP-6 on the 3rdday of fasting attenuated peak ghrelin levels in the afternoon. Thisclearly shows that fasting induces an acute and distinct diurnal rhythmin systemic ghrelin concentrations that is not present in the fed state.These changes in serum ghrelin levels during fasting are followed bysimilar changes in serum GH concentrations, indicating that ghrelin isthe driving force of increased GH secretion during fasting. Thisphenomenon cannot be explained by changes in insulin, glucose or freefatty acid levels. Thus it appears that the metabolic effects of ghrelinare distinct from its effects on GH secretion.

By the use of the GH receptor antagonist pegvisomant, indirect evidencewas provided that these changes in serum ghrelin levels are notregulated by the GH receptor. Finally, it was shown that administrationof the synthetic GHS, GHRP-6, produced a decrease in peak ghrelinlevels, but this effect was only observed after several hours,indicating the existence of a long-loop feedback system of GHS's onghrelin secretion.

In order to elucidate the metabolic effects of ghrelin, a study wasperformed on 11 healthy young male volunteers in whom glucose andinsulin levels were measured after a single intravenous administrationof human ghrelin (1.0 μg/kg i.v. at 0′) or placebo. FIG. 4 shows thatghrelin produced acute decrease in insulin [mean (±SEM) Δ insulin] (toppanel) and elevation in glucose [δ mean (±SEM) glucose] levels (bottompanel) (solid dots: ghrelin; open dots: placebo). This data clearlyshows that ghrelin has distinct and immediate effects on glucose andinsulin, two important determinants of metabolism in humans (Broglio Fet al. Journal of Clinical Endocrinology & Metabolism 2001;86(10):5083-5086).

Thus the data reported in the present application, indicate that toghrelin has important physiological actions, not only on GH secretionbut also on the modification of glucose and insulin concentrations inliving (human or animal) beings.

Ghrelin appears to have a role in managing not only GH secretion butalso the metabolic response to starvation by modulating insulinsecretion and glucose metabolism.

In an analysis of a study in normal human volunteers (n=6), it wassurprisingly observed that the administration of unacylated ghrelin (1μg/kg iv at 0 min) totally prevented the ghrelin (1 μg/kg iv at 0min)-induced increase in glucose and decrease in insulin levels. Thelots of unacylated ghrelin used in this study had the followingspecifications: tifluoroacetate salt of unacylated ghrelin, 95.1% pureas judged by HPLC, Mass: 3244.7 amu, and the peptide has amino acidcomposition representative of the sequence listed in SEQ ID NO: 1.

FIGS. 5A-5B show the mean (±SEM) Δ glucose (FIG. 5A) and Δ insulin (FIG.5B) levels after a single intravenous administration of human acylatedghrelin (1.0 μg/kg i.v. at 0′), human des-acylated ghrelin (1.0 μg/kgi.v. at 0′) or the co-administration of both. Thus it appears thatunacylated ghrelin is acting as a functional antagonist of theperipheral actions of ghrelin. This last result was surprising andunexpected, since unacylated ghrelin has never been shown previously toantagonize or inhibit the biological effects of acylated ghrelin. Mostof ghrelin actions, especially on GH secretion were thought to bemediated by GHS-R1a receptor for which unacylated ghrelin has littleaffinity. In fact, unacylated ghrelin has so far been considered as apeptide without any biological activity.

Hence in this invention, it is shown that unacylated ghrelin acts as afunctional antagonist to inhibit important peripheral actions ofacylated ghrelin on two crucial parameters of metabolism-insulin andglucose. To provide therapeutic benefits to patients in various statesof impaired glucose metabolism and/or insulin resistance, preferablythose associated with low GH action and/or increased acylated ghrelinsecretion, unacylated ghrelin(NH₂Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg:SEQ ID NO: 1) or its analogue may be administered in a pharmaceuticalcomposition intravenously, subcutaneously, transdermally, orally or byinhalation. Preparation of pharmaceutical compositions suitable forintravenous, subcutaneous, transdermal, oral, buccal, sublingual andpulmonary delivery are known to people skilled in the arts.

In this invention it is also demonstrated that unacylated ghrelin has adirect influence on glucose and on lipid metabolism, and on theproliferation and survival of beta cells.

In one aspect of the invention, the effects of unacylated ghrelin (1.0μg/Kg/h infused iv for 16 consecutive hours from 21.00 to 13.00 h) orsaline was evaluated in 8 healthy males (age mean±SEM:29.6±2.4 yrs;BMI:22.4±1.7 kg/m²) who had isocaloric balanced fixed meals at 21.20 and09.00 h. Glucose, insulin, glucagon, free fatty acids (FFA), GH, andcortisol were measured every 20 minutes.

Unacylated ghrelin infusion significantly modified the profile of allparameters, except glucagon. Compared to saline, unacylated ghrelindecreased free fatty acyl (FFA) and glucose AUCs (p<0.01) (FIGS. 6A to6D). The FFA profile was reduced both post-prandially (p<0.01) and atfasting (p<0.01), while glucose decrease during unacylated ghrelin wasparticularly relevant at fasting during night time (p<0.01) (FIGS. 6A to6D). Unacylated ghrelin did not modify total insulin AUC, which,altogether with the significant reduction in glucose levels, indicatedimproved insulin sensitivity; however the early insulin response to bothdinner (p<0.01) and breakfast (p<0.05) was enhanced by unacylatedghrelin (FIGS. 6E and 6F). During unacylated ghrelin infusion, cortisoland GH AUCs were lower (p<0.01) than those during saline, but cortisolremained within physiological levels) (FIGS. 6G and 6H). Since bothcortisol and growth hormone are hyperglycemic hormones, their reductionunder unacylated ghrelin infusion very likely also contributed to theobserved glycemia-lowering effect.

The intravenous infusion of unacylated ghrelin in normal subjectsenhances the early insulin response to meals, improves glucosemetabolism and insulin sensitivity, and decreases circulating free fattyacids levels. Thus, unacylated ghrelin displays a remarkable metabolicimpact, a promising anti-diabetogenic action through an originalmechanism of action.

Survival of pancreatic β-cells is obviously of major importance formaintaining normal glucose metabolism. Apoptosis of pancreatic β-cellsis a critical step in the development of type 1 diabetes, but β-cellgrowth and survival are critical also in type 2 diabetes. Inflammatorycytokines, including IFN-γ, TNF-α, and IL-1β are strongly implicated inpancreatic islet β-cell death and functional loss during autoimmunediabetes and also seem to be involved in early loss of islet mass inislet transplantation.

Unacylated ghrelin immunoreactivity was detected in HIT-T15 β-cells.Moreover, these cells were analyzed for their capacity of releasingunacylated ghrelin. ELISA experiments demonstrated that unacylatedghrelin was secreted by HIT-T15 cells (185 and 242 pg/ml, respectively)after 48 h incubation in complete medium. No expression of GRLN-R couldbe detected in HIT-T15 cells, either at the protein or at the mRNAlevel. HIT-T15 cells express ghrelin mRNA and peptide but not GRLN-R(not shown).

Experiments using increasing concentrations of ¹²⁵I-labeled[Tyr⁴]-unacylated ghrelin provided consistent evidence of a saturablespecific binding in HIT-T15 cells (FIG. 7). Scatchard analysis (notshown) demonstrated the existence of a single class of binding sitesthat showed values of B_(max) (13.9±0.8 fmol/mg protein) and Kd(0.68±0.10 nm, mean±sem of four independent experiments). Unlabeledunacylated ghrelin, as well as [Tyr⁴]-acylated ghrelin and hexarelin,but not somatostatin, insulin, or glucagon competed with ¹²⁵-labeled[Tyr⁴]-unacylated ghrelin for binding sites. Unacylated ghrelinrecognizes common high-affinity binding sites on HIT-T15 cell membranes.

Based on evidence of unacylated ghrelin-specific binding sites, theeffect of unacylated ghrelin on HIT-T15 cell proliferation wasinvestigated. In one variant of the invention, cells were incubated inserum-free medium in the presence or absence of increasingconcentrations, ranging from 1 pm to 1 μm (10⁻¹² to 10⁻⁶ m) ofunacylated ghrelin for 24 h, BrdU incorporation assay showed that thepeptide significantly and dose-dependently induced cell proliferation(FIGS. 8A and 8B). The efficacy of cell growth stimulation was within 1nm to 1 μm, equal to the one that was found effective in displacingradiolabeled unacylated ghrelin from HIT-T15 binding sites. This effectwas similar to that observed in cells cultured in normal conditions,i.e. in the presence of serum (15% HS, 2.5% FBS).

To investigate the signaling pathways involved in ghrelin mitogeniceffect, the cells were preincubated (30 min) with NF449, a selectiveGα_(s) protein-coupled receptor antagonist. This resulted in completeblockade of unacylated ghrelin-induced cell proliferation, whereaspretreatment with pertussis toxin (PTX; 50 ng/ml), an inhibitor ofGα_(i/o) protein coupled receptor, had no effect (FIGS. 8D-8E). FIG. 8Cis a representative phase contrast image showing that unacylated ghrelincounteracts HIT-T15 β-cell loss in serum deprived conditions byincreasing the number and size of islet-like structures, with respect tountreated cells. Taken together, these results show that unaclylatedghrelin promotes β-cell proliferation, likely involving the Gα_(s)signaling pathway.

In FIG. 8A to 8E, HIT-T15 cells were cultured in serum-free medium(Control) for 24 h, UAG alone or with NF449 and pertussis toxin (PTX)were added to the incubation medium for further 24 h. In FIG. 8A, cellproliferation was measured by BrdU uptake in cells cultured in thepresence or absence of serum, UAG at the concentrations indicated. InFIG. 8B, cell survival was measured by MTT in the presence or absence ofserum, IGF-I (15 nM), unacylated ghrelin at the concentrationsindicated. FIG. 8C illustrates a phase-contrast images of cellscultured±unacylated ghrelin (100 nM each). FIG. 8D and FIG. 8Eillustrate unacylated ghrelin proliferative and survival effect (100 nMeach), assessed by BrdU and MTT respectively, in the presence of NF499(10 μM) or pertussis toxin (PTX) (50 ng/ml), (C, control). Data areexpressed as the percentage relative to control and are the means±SEM ofeight replicates within a single representative experiment that wasrepeated at least 3 times (*P<0.05,**P<0.01).

Apoptosis is the main form of pancreatic β-cell death in animal modelsof type 1 diabetes mellitus. IFN-γ/TNF-α synergism has been shown toplay an important role in autoimmune diabetes in vivo as well as β-cellapoptosis in vitro. On the basis of the results showing that unacylatedghrelin promotes HIT-T15 cell proliferation, it was examined whetherunacylated ghrelin inhibited apoptosis induced by serum deprivation orby IFN-γ/TNF-α synergism. Hoechst 33258 staining showed that after 48 h,cells cultured in the presence of serum were round shaped, formedislet-like structures, and had very low apoptotic rate (˜2%) (FIG. 9Ainset, upper panel, and FIG. 9B). In serum-deprived medium, apoptosisincreased up to approximately 20%, and cells displayed typical chromatincondensation and nuclear fragmentation. Moreover, they partially losttheir capacity to form islet-like structures (FIG. 9A, upper panel, and9B). Cytokines further increased apoptosis (˜27%), cells appearingsmaller and unable to form islets (FIG. 9A, lower panel, and FIG. 9B).Although effects on glucagon and insulin release have been demonstratedwith ghrelin at low concentrations (<100 nm), on the basis of bindingstudies and cell proliferation results, 100 nm (10⁻⁷ m) was selected asthe preferred unacylated ghrelin concentration for the continuation ofthis study. Accordingly, others have reported that ghrelin exertsproliferative and antiapoptotic effects at high concentrations (100-1000nm) in different cell types. Unacylated ghrelin, preferably at 100 nm,almost completely prevented serum-starvation-induced apoptosis andrestored islet-like structures (FIG. 9A, upper panel, and FIG. 9B).Unacylated ghrelin significantly reduced apoptosis (˜12%) triggered bythe IFN-γ/TNF-α combination and induced cell enlargement and small isletformation (FIG. 9A, lower panel, and FIG. 9B).

The unacylated ghrelin antiapoptotic effect was dose dependent, 1 nm(10⁻⁹ m) being the lowest significantly active concentration of peptides(data not shown). Furthermore, caspase-3 activation in bothserum-starved and cytokine-treated cells was significantly reduced byunacylated ghrelin (data not shown) providing additional evidence of itsantiapoptotic effect in HIT-T15 pancreatic β-cells.

Indeed, cytokines strongly decreased cell proliferation, andunexpectedly, unacylated ghrelin dramatically restored cellproliferation up to rates that were even higher than those observed inthe presence of serum (FIG. 9C). Unacylated ghrelin effect on cellsurvival was also investigated by MTT assay in both serum-freeconditions and in the presence of cytokines. The results of theseexperiments indicated that unaclylated ghrelin significantly increasedcell viability under both experimental conditions (data not shown).

Herein, it was previously showed that HIT-T15 cells express and releaseunacylated ghrelin, indicating that it could act throughautocrine/paracrine mechanisms. To investigate this possibility,unacylated ghrelin secretion was measured in cells cultured in thepresence of serum and in serum-free medium alone or with addition ofIFN-γ/TNF-α. FIG. 9D shows that unaclylated ghrelin level wassignificantly reduced in serum-starved cells and even more afterexposure to cytokines. Surprisingly, addition of a specific antighrelinantibody with specificity for unacylated ghrelin not only increasedserum starvation-induced apoptosis but also induced apoptosis in cellscultured in the presence of serum, suggesting that endogenous unacylatedghrelin could exert autocrine/paracrine action on cell survival. Asexpected, no effect was observed in cytokine-induced apoptosis, whereunaclylated ghrelin secretion is likely too low to counteract such astrong cell death increase (FIG. 9E).

In FIGS. 9A to 9E, HIT-T15 cells were starved for 24 h and subsequentlyincubated for 24 h in the presence or absence of IFN-γ/TNF-α (100 ng/mland 200 ng/ml respectively), 100 nM unacylated ghrelin. FIG. 9Aillustrates a Hoechst 33258 nuclear immunofluorescence staining(magnification ×200) of serum starved cells±unacylated ghrelin (upperpanel; insert: cells with serum) and cells treated withIFN-γ/TNF-α±unacylated ghrelin (lower panel). In FIG. 9B, apoptosis isevaluated by counting condensed/fragmented Hoechst-stained nuclei (SF,serum-free medium). Values are expressed as percent of apoptotic cellsand are the mean±SEM of duplicate determinations (500 cells each) ofthree independent experiments (*P<0.05; **P<0.01). In FIG. 9C, cellproliferation is assessed by BrdU uptake (ELISA). The results areexpressed as percent of control (serum starved cells) and are themean±SEM of three independent experiments (*P<0.05,**P<0.01). FIG. 9Dillustrates ghrelin secretion in HIT-T15 conditioned medium followingexposure to either serum, SF±cytokines. The results are the mean±SEM ofthree independent experiments, each performed in quadruplicate(*P<0.05). In FIG. 9E, apoptosis is determined by Hoechst 33258 of cellscultured for 48 h in the presence of serum or in SF medium alone withaddition of an anti-ghrelin antibody (α-ghrelin Ab), (*P<0.05,**P<0.01vs 0 μg/ml α-ghrelin Ab in each condition).

Together, these results show that unaclyated ghrelin counteractsapoptosis induced by serum starvation and IFN-γ/TNF-α combination inHIT-T15 cells. Moreover, they strongly indicate that even endogenousunacylated ghrelin exerts cytoprotective effects, likely viaautocrine/paracrine mechanisms.

cAMP and its principal target, the cAMP-dependent PKA, play importantroles in mammalian cell proliferation and apoptosis. Elevation ofintracellular cAMP levels has been shown to promote cell growth and todelay apoptosis in different cell types, including pancreatic β-cells.Previous results showed that ghrelin-induced HIT-T15 cell proliferationinvolves the Gα_(s) protein-coupled receptor, which, in turn, has beenshown able to activate cAMP/PKA signaling; therefore, it wasinvestigated whether the proliferative and antiapoptotic effects ofunacylated ghrelin is mediated by this pathway.

Initially, the unacylated ghrelin-induced cAMP intracellular variationwas examined. FIG. 10A shows that incubation of HIT-T15 cells with theunacylated ghrelin peptide, in the presence of the phosphodiesteraseinhibitor IBMX, resulted in time-dependent changes of cAMP levels.Unacylated ghrelin produced a transient increase within 5 min, which waslower but still significantly above basal level at 10 and 30 min,declining thereafter toward the resting level after 60 min incubation.

cAMP induction by ghrelin was then evaluated at 15 min in eitherserum-free medium alone or with addition of IFN-γ/TNF-α in the presenceof IBMX. Results showed that unacylated ghrelin significantlyup-regulated cAMP not only in serum-free medium alone but also afterincubation with cytokines that, per se, reduced cAMP levels (FIG. 10B).

FIGS. 10A and 10B demonstrate the effect of unacylated ghrelin onintracellular cAMP concentration in HIT-T15 cells. In FIG. 10A, serumstarved cells were cultured for the indicated times with 100 nM ofunacylated ghrelin. The results are the mean±SEM of three independentexperiments performed in triplicate (*P<0.05 vs basal time point). FIG.10B illustrates the cAMP levels in cells incubated in the presence ofserum or in serum-free (SF) medium±unacylated ghrlein (100 nM each)alone or with IFN-γ/TNF-α combination (100 ng/ml and 200 ng/mlrespectively). Data are the mean±SEM of at least three independentexperiments performed in triplicate (*P<0.05).

In this invention, it is demonstrated that unacylated ghrelin has aglucose lowering effect since unacylated ghrelin prevents thehyperglycemic effects of acylated ghrelin. The results presented hereinalso indicate that unacylated ghrelin has an insulin sensitizing effect,reduces cortisol and growth hormone levels, and reduces glycemia. Theresults in healthy volunteers clearly showed that unacylated ghrelinalone is able to reduce blood glucose levels and improve insulinsensitivity.

The data presented herein also demonstrate that unacylated ghrelindecreases free fatty acids (FFA) in blood, indicating an effect ofunacylated ghrelin on dyslipidemia. It might be expected that moreprolonged treatments with unacylated ghrelin will have an effect onother lipids, such as, but not limited to triglycerides. In addition tothese properties, unacylated ghrelin is capable of stimulating theproliferation and the survival as well as inhibiting death ofinsulin-secreting cells such as, but not limited to, pancreatic β cells.

In another aspect, the present invention provides for applications ofunacylated ghrelin in the reduction, treatment and prevention ofdiseases, disorders and/or conditions associated with impaired glucose,insulin and lipid metabolism. The present invention also provides for anapplication of unacylated ghrelin and its analogs in modulating theproliferation of insulin-secreting cells. Such disorders and/orconditions include, but not limited to, type I diabetes, type IIdiabetes, the metabolic syndrome, dyslipidemia, and any medicalconditions associated with insulin resistance. Unacylated ghrelin or itsanalogs can also be used to improve the quality of beta islets graftsprior to engraftment, and also improve the survival of beta cells in apatient following engraftement.

Hence, in this invention it was shown that unacylated ghrelin acts as ananti-diabetogenic agent with applications in the prevention andtreatment of metabolic disorders associated with the metabolic syndrome,or syndrome X. Indeed, the demonstrated action of unacylated ghrelin toreduce blood glucose levels, to improve insulin sensitivity, to decreaseblood free fatty acids (FFA) and cortisol levels, as well as its actionto promote proliferation of insulin-secreting pancreatic β-cells areindicative of this anti-diabetogenic effect.

In accordance with a further aspect of the invention, therapeuticcompositions of the present invention, comprising a therapeuticallyeffective amount of an agent selected from the group consisting of anunacylated ghrelin, an analog thereof and a pharmaceutically acceptablesalt thereof, may be provided in containers or commercial packages whichfurther comprise instructions for use of a therapeutically effectiveamount of an agent selected from the group consisting of an unacylatedghrelin, an analog thereof and a pharmaceutically acceptable saltthereof for the prevention and/or treatment of diseases.

Accordingly, the invention further provides a commercial packagecomprising a therapeutically effective amount of an agent selected fromthe group consisting of an unacylated ghrelin, an analog thereof and apharmaceutically acceptable salt thereof or the above-mentionedcomposition together with instructions for the prevention and/ortreatment of diseases.

It is understood that the data reported in the present specification areonly given to illustrate the invention and may not be regarded asconstituting a limitation thereof.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A method for preventing and/or treating a metabolic disorderassociated with impaired glucose metabolism in a patient comprisingenhancing survival of insulin-secreting cells ex vivo by subjecting theinsulin-secreting cells to a therapeutically effective amount of anagent selected from the group consisting of an unacylated ghrelin, ananalog thereof and a pharmaceutically acceptable salt thereof, to saidpatient, prior to administering said cells to the patient as a graft. 2.A method for enhancing survival and/or proliferation ofinsulin-secreting cells comprising culturing said cells in the presenceof a therapeutically effective amount of an agent selected from thegroup consisting of unacylated ghrelin and an analog thereof.
 3. Themethod of claim 2, wherein unacylated ghrelin consists of the amino acidsequence set forth in SEQ ID NO: 1 or an analog thereof.
 4. The methodof claim 2, wherein the insulin-secreting cells are pancreatic β-cells.5. A method for inhibiting death of insulin-secreting cells comprisingculturing said cells in the presence of a therapeutically effectiveamount of an agent selected from the group consisting of unacylatedghrelin and an analog thereof.
 6. The method of claim 5, whereinunacylated ghrelin consists of the amino acid sequence set forth in SEQID NO: 1 or an analog thereof.
 7. The method of claim 5, wherein theinsulin-secreting cells are pancreatic β-cells.